Atp8b1 deficiency in mice reduces resistance of the canalicular membrane to hydrophobic bile salts and impairs bile salt transport.

Progressive familial intrahepatic cholestasis type 1 (PFIC1, Byler disease, OMIM 211600) is a severe inherited liver disease caused by mutations in ATP8B1. ATP8B1 is a member of the type 4 subfamily of P-type ATPases, which are phospholipid flippases. PFIC1 patients generally develop end-stage liver disease before the second decade of life. The disease is characterized by impaired biliary bile salt excretion, but the mechanism whereby impaired ATP8B1 function results in cholestasis is unclear. In a mouse model for PFIC1, we observed decreased resistance of the hepatocanalicular membrane to hydrophobic bile salts as evidenced by enhanced biliary recovery of phosphatidylserine, cholesterol, and ectoenzymes. In liver specimens from PFIC1 patients, but not in those from control subjects, ectoenzyme expression at the canalicular membrane was markedly deficient. In isolated mouse livers Atp8b1 deficiency impaired the transport of hydrophobic bile salts into bile. In conclusion, our study shows that Atp8b1 deficiency causes loss of canalicular phospholipid membrane asymmetry that in turn renders the canalicular membrane less resistant toward hydrophobic bile salts. The loss of phospholipid asymmetry may subsequently impair bile salt transport and cause cholestasis.

Annexin V imaging of acute doxorubicin cardiotoxicity (apoptosis) in rats.

UNLABELLED: Anthracyclines are widely used in chemotherapy regimens for several malignancies, with cardiotoxicity being the major limiting factor in high-dose schedules. Recently, it was reported that doxorubicin induces apoptosis in cardiac muscle cells in vivo and, as such, is expected to be involved in the genesis of doxorubicin-induced cardiomyopathy. The aim of this study was to validate an animal model for in vivo monitoring of doxorubicin cardiotoxicity by means of scintigraphic detection of apoptosis. METHODS: Three groups of 5 male Wistar rats each were treated for 3, 4, and 5 times with a weekly intraperitoneal injection of doxorubicin at 2.5 mg/kg. At 24 h before and 24 h after the final treatment, (99m)Tc-annexin pinhole scintigraphy was performed. A control group of 5 rats was scanned without doxorubicin treatment. A cardiac uptake ratio was calculated from planar scintigraphy results with the following formula: (mediastinum - fat)/fat. After scintigraphy, the rats were sacrificed, and the heart was processed for histologic analysis. RESULTS: Incremental general signs of illness were observed with increasing total cumulative doxorubicin dose. Rats treated for 3, 4, and 5 wk with doxorubicin showed significantly higher uptake ratios of, respectively, 4.0 +/- 0.52 (mean +/- SEM), 4.8 +/- 0.46, and 5.2 +/- 0.17 after the final treatment; the ratio for controls was 1.84 +/- 0.05 (P < 0.05). Histologic analysis confirmed cardiac stress in treated groups, with an increasing left ventricular atrial natriuretic factor messenger RNA expression level with increasing cumulative doxorubicin dose. Late apoptosis was confirmed by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling in the rats treated for 5 wk. CONCLUSION: Acute doxorubicin-induced cardiomyopathy based on early apoptosis can be assessed and imaged with annexin V scintigraphy in rats. This finding makes it possible to use this animal model for repetitive noninvasive evaluation of cardioprotective regimens for anthracycline cardiotoxicity.

Validation of gastric-emptying scintigraphy of solids and liquids in mice using dedicated animal pinhole scintigraphy.

UNLABELLED: Gastric emptying in small laboratory animals is a useful parameter to assess gastrointestinal motility for physiologic, pharmacologic, or other research purposes. In mice, phenol red recovery is considered the gold standard for determination of gastric emptying. However, this method requires sacrifice of the animal and yields data of gastric emptying at only 1 time point. Gastric-emptying scintigraphy, the gold standard technique in humans, allows sequential and serial measurements in the same subject. In this study, we developed and validated a novel method of gastric-emptying scintigraphy applied in mice, by comparing it with phenol red photospectrometry. METHODS: A dedicated animal pinhole gamma camera was equipped with a specially designed mouse application device. Gastric emptying was measured in unanesthetized mice using pinhole scintigraphy. First, gastric emptying determined with scintigraphy was compared with gastric phenol red recovery simultaneously within the same population. Subsequently, normal values for gastric emptying of solids and liquids in mice were established, and finally, the effects of handling stress and the late effects of frequently used anesthetics or sedatives on gastric emptying were assessed by scintigraphy. RESULTS: Gastric emptying of liquids measured by pinhole scintigraphy did not significantly differ from that measured by phenol red recovery. For the same information, 80% fewer animals were needed for the scintigraphic method. More stress-related delay in gastric emptying was induced by multiple handling of the mice, compared with the less frequent handling that was associated with taking measurements every 10 min or more (P < 0.05). The mean half-emptying time for solids measured by scintigraphy was significantly slower than that for liquid emptying (P < 0.01). Previous anesthesia did not significantly affect gastric emptying 6 h after induction. CONCLUSION: Dedicated small-animal pinhole gastric-emptying scintigraphy is a reliable tool to investigate gastrointestinal motility in mice, significantly reducing the number of laboratory animals needed for statistical power in trials. The technique enables sequential and serial measurement within 1 subject and is thus useful for follow-up investigations, which can be performed even after invasive procedures that require anesthesia.